The complement system of humans and other mammals involves more than 20 components which participate in an orderly sequence of reactions resulting in complement activation. Numerous studies indicate that the complement system is a fundamental element of normal host defense mechanisms. As a consequence, complement activation is commonly associated with a variety of pathological states such as certain malignancies, myocardial infarction, systemic lupus erythematosis, and adult respiratory distress syndrome. Because of these correlations clinical laboratory methods that detect complement activation are useful in diagnosing certain disease conditions.
Complement activation can occur by either of two primary modes known as the "classical" pathway and the "alternative" pathway, respectively. These different pathways are generally distinguished according to the process which initiates complement activation. Activation via the classical pathway is usually associated with an immunologic stimulus whereas activation via the alternative pathway is most commonly associated with non-immunologic stimuli. Regardless of the initiating stimulus both pathways converge, followed by the conversion of the C.sub.3 component of complement into its C.sub.3 a and C.sub.3 b fragments. This cleavage of C.sub.3 into its subcomponents its considered to be one of the significant events signalling activation of the alternate complement cascade. Following the conversion of C.sub.3, a C.sub.5 convertase enzyme complex is formed. This enzyme cleaves the C.sub.5 component to yield the fragments C.sub.5 a and C.sub.5 b. Complement activation by the classical pathway mechanism is uniquely characterized by the fact that this route leads to the conversion of the C.sub.4 component to its fragments C.sub.4 a and C.sub.4 b.
The physicochemical and physiological properties of the cleavage products C.sub.3 a, C.sub.4 a and C.sub.5 a, termed anaphylatoxins are well known. Each is a potent bioactive polypeptide and plays a key role as a mediator of acute inflammatory processes. Among these three anaphylatoxins C.sub.5 a alone is uniquely characterized by its ability to interact with white blood cells. Both C.sub.3 a and C.sub.4 a are rendered inactive in vivo by conversion of their respective des arginine derivatives (C.sub.3 a.sub.des Arg or C.sub.3 a.sub.i, C.sub.4 a.sub.des Arg or C.sub.4 a.sub.i) by a serum carboxypeptidase. Human C.sub.5 a, on the other hand, is converted to C.sub.5 a.sub.des Arg by this serum carboxypeptidase only after all available white blood cell binding sites for C.sub.5 a have been saturated.
Conversion of the human complement components C.sub.3 and C.sub.5 to yield their respective anaphylatoxin products has been implicated in certain naturally occurring pathologic states including: autoimmune disorders such as systemic lupus erythematosis, rheumatoid arthritis, malignancy, myocardial infarction, Purtscher's retinopathy, and adult respiratory distress syndrome. In addition, increased circulating levels of C.sub.3 a and C.sub.5 a have been detected in certain conditions associated with iatrogenic complement activation such as: cardiopulmonary bypass surgery, renal dialysis, and nylon fiber leukaphoresis. Elevated levels of C.sub.4 a anaphylatoxin are commonly associated with the autoimmune disorders mentioned above. Therefore, the ability to quantitatively measure the circulating levels of these anaphylatoxins or their des-Arg derivatives is of great utility in diagnosing a variety of important pathological conditions. Additionally, the ability to measure levels of C.sub.4 a or C.sub.4 a.sub.des Arg enables one to determine the pathway by which complement activation occurs. This facility enables one not only to determine the precise mechanism of complement activation but also whether a patient's natural immunological defense mechanisms are functional.
Until the development of the radioimmunoassay (RIA) method of Tony E. Hugli and Dennis E. Chenoweth reported in "Immunoassays: Clinical Laboratory Techniques for the 1980s," 443-460, Alan R. Liss, Inc., New York, NY (1980), measurement of the anaphylatoxins C.sub.3 a, C.sub.4 a and C.sub.5 a or their des-Arg derivatives had only been achieved when the levels of these factors were relatively elevated, for example, when the disease process had reched an advanced stage. The RIA techniques of Hugli and Chenoweth permit quantitative measurement of trace amounts of the anaphylatoxins or their des-Arg derivatives and hence provide a sensitive diagnostic tool. However, the means known heretofore for measuring these factors have been frought with a significant problem associated with the requirement that the C.sub.3, C.sub.4 and C.sub.5 plasma precursors of the anaphylatoxins must be removed from the biological fluid to be tested. This stringent requirement is predicated on the observation that the antibodies raised to the anaphylatoxins possess a significant cross-reactivity with their respective plasma precursor as C.sub.3 a, C.sub.4 a or C.sub.5 a is a part of the parent molecule C.sub.3, C.sub.4 and C.sub.5, respectively. Because of this unavoidable cross-reactivity it is imperative that the precursor which exists in relatively high concentrations in serum and plasma be completely removed to avoid detecting artifactually elevated levels of the anaphylatoxins which are normally present in only trace amounts. Prior known methods of separating the anaphylatoxins from their plasma precursors involves diluting the plasms with sodium chloride and acidifying with hydrochloric acid and subsequently neutralizing the recovered serum sample. See Hugli and Chenoweth cited above. The present invention provides a novel and simplified means of quantitatively removing the plasma precursor of the anaphylatoxin from the biological samples yet simultaneously permitting a quantitative recovery of the low molecular weight anaphylatoxins, C.sub.3 a, C.sub.4 a and C.sub.5 a or the des-Arg derivatives thereof.